Inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) with different multi-locus sequence types (MLST), SCCmec, and spa types

Background The inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) are unclear. The main aim is to better understand of inhibitory activities of vitamins K2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutation and gene expressions. Materials and methods After collecting S. aureus clinical isolates and detecting QR-MRSA, the genes encoding norA, grlA, grlB, gyrA, and gyrB were sequenced. After treating isolates by vitamin K2, isolates were prepared to measure norA, grlA, grlB, gyrA, and gyrB gene expression. The quantitative-real-time PCR was used to measure the expression of efflux pump genes. Results QR-MRSA, MDR, and XDR strains were reported in 59.4%, 73.9%, and 37.6% of isolates, respectability. SCCmecIV (36.5%) and SCCmecV (26.8%) had the highest frequency. Thirty-nine spa types were identified, t021, t044, and t267 types most prevalent in QR-MRSA isolates. ST22 and ST30 dominated the invasive, drug-resistant isolates and QR-MRSA. In 24 h incubated isolates, the most noticeable change of gene expression with vitamin K2 was that the norA, gyrA, and grlB genes were highly repressed. However, the down-regulation of grlA at 24 h after being treated by vitamin K2 was more than another gene. Further, a significant decrease was observed in QR-MRSA-treated isolates compared to un-treated isolates. In other words, norA, grlA, grlB, gyrA, and gyrB genes were less suppressed by QR-MRSA (p ≤ 0.01, p ≤ 0.05). Conclusion Vitamin K2 has significant inhibitory effects on the genes responsible for resistance to fluoroquinolone antibiotics. However, a subminimum inhibitory concentration (sub-MIC) level of vitamin K2 was delayed but did not completely inhibit norA, grlA, grlB, gyrA, and gyrB genes in MRSA strains.

Ciprofloxacin and ofloxacin were the most extensively used fluoroquinolones to treat quinolone-resistant and methicillin-resistant S. aureus (QR-MRSA) [5]. Resistance to this class of agents occurs by two main processes. The first one, caused by mutations in the target enzymes, lowers the drug's affinity for the DNA topoisomerase complex [5]. Those mutations occur in the cellular targets GyrA/GyrB of DNA gyrase, encoded by genes gyrA/gyrB and GrlA/GrlB of topoisomerase IV, encoded by genes grlA/grlB [5][6][7]. The second one, caused by overexpression of efflux pumps. Although the function and composition of MDR efflux pumps are relatively conserved in different species, their regulatory mechanisms vary significantly [5,8].
With the increasing utilization of fluoroquinolones to fight against QR-MRSA, emerging resistance to these agents is growing. Available treatments for QR-MRSA infections are expanding chemical compounds such as herbal extracts, mineral composition, and vitamins [8-10].
Vitamin K (K 1 , K 2 , K 3 ) plays an essential role in blood coagulation and protein synthesis processes in plasma, kidneys, and other tissues [11]. Moreover, the inhibitory effects of vitamin K 2 on various neoplastic cells and reduced risk of mutagenic events in rapid cell proliferation in the fetus and newborn were reported [12,13]. Nevertheless, the modulation of plasma membrane permeability by lipid-soluble compounds was reported. The precise mechanism of these vitamins on the resistance factors associated with QR-MRSA has not been studied [9,10,14].
The effect of DNA gyrase and topoisomerase mutations and gene expressions on minimum inhibitory concentrations (MICs) was studied to understand better inhibitory activities of vitamins K 2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutations and gene expressions.
Thus, this study aimed to determine the effect of DNA gyrase and topoisomerase mutations on minimum inhibitory concentrations (MICs) of vitamins K 2. This purpose was destined for a better understanding of inhibitory activities of vitamins K 2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutation and gene expressions.

Design of study and bacterial isolates
In this study, the isolates were collected between June 2019 and August 2020 from 460 clinical samples by diagnostic microbiology laboratories. Isolates were collected from specimens such as pus swabs (ear, nose and eye, cervical and wound), catheter tips, sputum, blood, body fluids, urine, and CSF throughout Hamadan hospitals.
Morphological and biochemical testing was performed to confirm S. aureus. For confirmation of S. aureus isolates, white colonies surrounded by halos from Blood agar (Hi-Media, India) with 5% sheep blood after incubation for 24 h at 37 °C were plated onto mannitol salt agar (Hi-Media, India). Reaction on mannitol salt agar was interpreted and recorded as positive or negative based on criteria described by Mahon et al. [15]. Then, conventional identification methods were used, which included colony morphology, mannitol fermentation, catalase reaction, and coagulase reaction. Finally, 69 isolates of S. aureus were collected from different specimens.

Antibiotic resistance profile and MRSA strains
Antimicrobial resistance tests were performed using the standard Kirby Bauer disk diffusion method as recommended by Clinical Laboratory Standards Institute [16]. Antibiotics were selected from different categories, containing gentamicin (10 μg), erythromycin (15 μg), tetracycline (30 μg), ciprofloxacin (5 μg), gatifloxacin (5 μg), norfloxacin (10 μg), ofloxacin (5 μg), rifampin (5 μg), penicillin (10 unit), clindamycin (2 μg), and linezolid (30 μg). All antibiotic disks belonged to the MAST Company (MAST Inc., U.K.). For detection of MRSA strains, S. aureus isolates were subjected to cefoxitin (30 µg) (MAST Inc., U.K.) sensitivity test by the Kirby Bauer disk diffusion method. Isolates resistant to at least one agent in three or more antimicrobial classes were identified as multidrug-resistant (MDR). Isolates resistant to at least one agent in all but two or fewer antimicrobial classes were considered extensively drug-resistant (XDR). Isolates with non-susceptibility to all agents in all antimicrobial classes were referred to as pan drug-resistant (PDR) [17]. S. aureus ATCC 25923 strain was used as quality control.

Minimum inhibitory concentration (MIC) of ciprofloxacin and vitamin K 2
Using an E-test strip (Liofilchem, Italy), minimum inhibitory concentration (MIC) of ciprofloxacin was detected in all isolates. Also, to determine the antibacterial properties of vitamin K 2 , the microdilution method was used. In this method, MIC was determined based on the method described by Tintino et al. [13]. S. aureus ATCC 25923 strain was used as quality control.

SCCmec and spa typing
SCCmec and spa typing were carried out according to Vafaeefar et al. and Goudarzi et al. [18,19]. SCCmec I, II, III, IV, and V types were determined based on the amplification pattern obtained. Cluster analysis of spa types was performed using the Ridom Staph Type version 2.2.1 (Ridom GmbH, Würzburg, Germany), a built-in feature of the Staph Type software [20].

Multi-locus sequence typing (MLST)
The  [21]. Sequence alignments were performed using Clustal W with default parameters. All columns in the multiple alignment matrix with more than 80% gaps were eliminated.

Statistical analysis
All statistical analyses were performed using the Graph-Pad Prism software (version 5; GraphPad Software Inc.; La Jolla, CA, USA. The Chi-square statistical (p) test and Pearson's correlation coefficient (r) were chosen to explore the association between categorical variables. The difference between the amounts of antibiotic resistance and the prevalence of genes in the various media was statistically significant when p < 0.05.
The relative expression levels of the genes (at 6, 12, and 24 h.), compared to calibrator at 0 h incubation, were normalized and determined from the expression of the reference gene. Expression levels of the genes utilizing the ∆∆Ct method (the target gene = 2 −ΔΔCq (where ΔCq = Cq (target gene) -Cq (reference gene) , and ΔΔCq = ΔCq (test) -ΔCq (calibrator) ). The primer efficiency calculations were determined utilizing REST software version 2008 as described by Pfaffl et al. [22,23]. All statistical analyses were performed using the GraphPad Prism software (version 5; GraphPad Software Inc.; La Jolla, CA, USA), and the Student's T-test (two-tailed and two-sample) was carried out. The Cq value of the reference gene and the stability of expression were analyzed using a T-test, twoway ANOVA, and Wilcoxon signed-rank test. A variation with a p < 0.05 was considered statistically significant.

MIC of ciprofloxacin and vitamin K 2
According to Fig. 2, 42 isolates (60.8%) with a 4 μg/ml MIC and ciprofloxacin (Fig. 2a) resistance were considered. Also, nine isolates were sensitive to vitamin K 2 (Fig. 2b), 21 isolates were intermediate, and others were resistant to vitamin K 2 . All MRSA strains were entirely resistant to vitamin K 2 .

Measurement of norA, grlA, grlB, gyrA, and gyrB genes activity
This experiment revealed that vitamin K 2 decreased the expressions of norA, grlA, and grlB genes by 30, 54and 21-fold, respectively, compared to the un-treated isolates. In contrast, the addition of vitamin K 2 significantly induced the expression of gyrB, which was down-regulated only by 18-and 12-fold, respectively, relative to the control. All results are shown in Figs. 3 and 4. According to Fig. 5, high-expression of gyrA, grlA, and grlB genes was observed in S. aureus isolated from wound and urinary tract infections. Despite this, efflux pump gene expression in bacteria isolated from blood and pus swab showed low-expressions levels.

Statistical analysis results
The statistical analyses of the present study are shown in Table 1. Comparing both un-treated and treated isolates showed a significant correlation between vitamin K 2 and antibiotic resistance patterns. In other words, the MIC of vitamin K 2 showed a significant difference in MDR, XDR, and antibiotic-sensitive isolates (p < 0.05). Based on χ 2 and t-test, a significant association was reported between MIC of vitamin K 2 and SCCmec type. Further, a strong correlation between the prevalence of norA, grlA, grlB, gyrA, and gyrB genes and MIC of vitamin K 2 was observed (p < 0.05) (p < 0.001). However, a negative correlation between spa typing and MIC of vitamin K 2 was observed in this study (p > 0.05). Based on Figs. 4 and 5, a significant correlation was reported between the expression of norA, grlA, grlB, gyrA, and gyrB genes and resistance to the antibiotic. A clear correlation was observed between norA, grlA, grlB, gyrA, and gyrB genes expression SCCmec typing. Thus, the expression of norA, grlA, grlB, gyrA, and gyrB genes showed a significant decrease in MRSA and MSSA strains (p > 0.001). Still, no good correlation was observed between norA, grlA, grlB, gyrA, gyrB expression levels, and spa typing (p > 0.001). Also, Table 2 and Fig. 5 show a significant association between the clinical specimens and the expression of efflux pump genes.

Discussion
Among the 69 S. aureus isolates obtained in this study, 30.4% were collected from blood, 24.6% from urine, 23.1% from the burned wound, 13% from pus swap, and 8.6% from the catheter. In a study conducted by Kot et al. [24], a high prevalence of blood and wound infection by S. aureus was reported. Most studies have demonstrated that there is a significant association between antibiotic resistance patterns and clinical specimens. In wound infections, the bacteria are more resistant to treatment, consistent with the current study [2,8].
Most isolates were resistant to penicillin (75.3%) and ciprofloxacin (62.3%). Also, 52.1% and 20.2% of the isolate were MDR and XDR. This observation agrees with Cabrera et al. and Kot et al., similar to that investigated in this study [24,25]. These findings are in contrast to the data reported from Singapore [26], Nepal [27], and the United States [28].
In the present study, based on QR-MRSA MIC, 2409C, T2460G, T1497C most common mutation in norA, grlA, grlB, gyrA, and gyrB genes. The observations also agree with the results reported by Hassanzadeh et al. [29] and Hashem et al. [30]. SCCmec typing for MRSA isolates showed the predominance of SCCmec type IV (63.4%) followed by type V (56%), type II (53.6%), type III (46.3%), and type I (31.7%). A similar pattern of results was observed in the study of Taherikalani et al. [31]. Moreover, these results were essentially confirmed by some studies from Saudi Arabia [32], Iraq [1], and South Africa [33], which stated that SCCmec IV and V were the most dominant types.
However, spa typing in the present study indicated that t030, t044, and t037 were the most common types, and t267 was a unique type in MRSA strains. In this study, the diversity of spa types in MRSA was more extensive than previously found in S. aureus in Iran [34,35]. A high prevalence of t044 (31.7%) was detected in MRSA. This finding was also reported in Kuwait and [36], Iran [37] as well as in Europe [38,39]. On the other hand, in the MSSA strains, t044, t037, and t030 were the most prevalent spa-types, which was not comparable with the findings of Satta et al. [40] and Mazi et al. [41].
Quantitative real-time PCR results showed that norA, grlA, and grlB genes were down-regulated in MSSA after treatment with vitamin K 2 . Generally, in MRSA strains, at 6 h, norA, grlA, were down-regulated; at 12 h, it was up-regulated, and at 24 h, it was down-regulated. Surprisingly after treating MRSA and MSSA with vitamin K2, the grlB and gyrA gene was up-regulated at 6 h and down-regulated at 12 h (−2.737). In MDR/MRSA strains, the gyrB gene was also up-regulated at 12 h and downregulated at 24 h (−0.737). This study's results seem to correlate with Tintino et al. [13], where norA was downregulated at 12 h. The results obtained here may have implications for understanding that methicillin resistance plays a critical role in the function of vitamin K 2 . However, it can be said that MRSA strains showed more significant changes in the resistance due to the effect of vitamin K 2 .
According to Table 1 and Fig. 5, the effect of vitamin K 2 also showed a significant difference in the clinical specimen types. Different changes in norA, grlA, grlB, and gyrA activity gene expression were obtained in strains isolated from urine culture. The most crucial reason for the difference in vitamin K 2 effect on clinical isolates is the typical and high consumption of fluoroquinolones to cure urinary tract infection infections. Hence, special attention should be paid to the isolates' source to inhibit efflux pumps in staphylococcal infections. We found agreement when comparing our   observations with results from Brazil [14,42], Germany [12], and Norway. Our study's findings show that gene expression increases significantly in blood isolates than in urine and wound isolates; however, some strains are susceptible to fluoroquinolones. Therefore, some studies have demonstrated some effectors on efflux pumps, such as the type of clinical specimens, which should be considered in clinical and laboratory investigations [9, 11, 43]. Tintino et al. [14] and Harakeh et al. [44] confirmed that some fat-soluble vitamins could increase antibiotic penetration in drug-resistant strains. They also suggested that some natural vitamins have a high effect on reducing the activity of β-lactamase enzymes. However, our result indicated the vitamin K 2 significantly down-regulated norA, grlA, grlB, gyrA genes giving fold changes of -1.022, -2.611, -1.891, -1.936, and -3.442 at 6, 12, and 24 h. On the other hand, the expression pattern of norA, grlA, grlB, gyrA genes in both MRSA and MSSA was completely different from the control sample.
The current study, Fig. 3, shows that norA, grlA, grlB, and gyrA were down-regulated in SCCmec type IV, V, and III after treatment with vitamin K 2 . Also, the pattern of expression of norA, grlA, and gyrA in isolates carrying SCCmec IV and V appears very different from that of isolates carrying SCCmec I and II genes. However, in the expression pattern observed before treatment of isolates, norA, grlA, grlB, gyrA genes in all SCCmec types at 24 h were down-regulated. Although similar studies in norA, grlA, grlB, gyrA activity are not available, Choi et al. [45], Yuan et al.
[10], and Qu et al. [46] showed the antimicrobial effect of vitamin K on MRSA strains. They also found that vitamin K had different functions in MRSA and drug-sensitive strains. These findings provide further evidence that, during the stationary phase, expression of grlA, grlB, and gyrA decreased by sevenfold in treated MSSA strains in laboratory conditions, independent of the vitamin k2, in fluoroquinolones resistance. This suggested additional regulatory mechanisms for this resistance [9].
Consistent with the findings of other studies [40,41], we found that t037 and t044 are the essential spa types in MRSA, MDR, and XDR strains. Thus, it was further observed that after the vitamin K 2 treatment of S. aureus with t037 and t044 spa typing, all genes except the gyrB were down-regulated in the strains giving fold changes of −1.120 (p = 0.01), −2.690, −1.999 (p = 0.02) and −0.120, −0.152 and 0.251 (p = 0.22) after 12 and 24 h. The grlA, grlB, and gyrA genes were also down-regulated in response to vitamin K 2 , with an increased expression between 3.0 and 4.2 log2-fold in isolates with spa typing t021, t267, and t030. The norA gene was also significantly down-regulated with a 2.2 log2-fold change in t267 and t030 types.
Finally, our knowledge from the present study confirmed the inhibitory effect of vitamin K 2 on the S. aureus efflux pump. Previous studies showed the inhibitory function of vitamin K 2 on the norA gene (14). However, we found that fat-soluble vitamins are among the best options for inhibiting fluoroquinolone efflux pumps genes (grlA, grlB, and gyrA) in S. aureus. In the function of vitamin K 2 on different strains of S. aureus, special attention should be paid to the type of clinical specimen and drug resistance. Another important factor that accounts for the survival of such mutants in the environment is microbial fitness. NorA-mediated resistance has been described in the apparent absence of mutations in topoisomerase genes. Indigenous microbial populations are made up of different communities of the same bacteria, which co-exist and compete for nutrition, space, and growth factors.

Conclusions
Based on the evidence obtained from the present study, vitamin K 2 had a good effect in inhibiting puffy pumps' activity in S. aureus. It was also found that the function of vitamin K 2 is significantly different in MRSA and MSSA strains. Significant differences in the expression of norA, grlA, grlB, and gyrA in different SCCmec types identified this locus's role in the function of vitamin K 2 . We also found that vitamin K 2 was a good option for inhibiting MDR and XDR strains. It should be noted that different types of SCCmec, antibiotic resistance patterns, and clinical specimen types are the essential variables in the treatment of clinical isolates of S. aureus with vitamin K 2 .